Figure 3. Mael depletion induces spontaneous DNA damage and ROS production in cancer cell lines.

A. Cell-cycle progression, monitored 72 hours after transfection, is depicted as a histogram of DNA content. B, C. The effects of Mael depletion on DNA damage were analyzed by immunofluorescence using an anti-γH2AX antibody and by western blotting using DNA damage-related antibodies. The protein lysates from HeLa cells were used for western blot analysis. D. Western blot detection of PARP cleavage in HeLa cells transfected with siRNAs selectively targeting specific Mael isoforms. E. Intracellular ROS levels in Mael-depleted cancer cells and normal cells, determined by measuring DCFDA fluorescence, were analyzed by flow cytometry and confocal microscopy. The FACS histogram of DCFDA intensity is a representative of three experiments. F. ROS levels in each experiment were quantified by measuring DCFDA mean fluorescence. The result was acquired from the average of three independent measurements. Statistical significance was determined by Student's t-test. G. intracellular ROS levels were visualized by microscopy.